Several research articles and patent documents are cited throughout this application to more fully describe the state of the art to which this invention pertains. The entire disclosure of these citations is incorporated by reference herein as though set forth in full.
Cytochromes P450 are the principal enzymes for the oxidative metabolism of drugs and other xenobiotics. Among the cytochromes P450, five forms, CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, appear to be most commonly responsible for the metabolism of drugs (1). Inhibition or induction of cytochrome P450-mediated metabolism is often the underlying mechanism responsible for drug-drug interactions (2). Such interactions can lead to a substantial decrease or increase in the blood and tissue concentrations of a drug or metabolite. These types of changes can alter a new drug's safety and efficacy profile in important ways, particularly those drugs with a narrow therapeutic range.
The development of sensitive and specific assays for a drug and its metabolite(s) is critical to the study of the drug's metabolism and interactions. For most drugs the metabolizing enzyme(s) is (are) known and chemical inhibitors for the specific enzyme(s) are available. The potential for enzyme inhibition is routinely assessed by performing in vitro inhibition studies using cDNA-expressed enzymes or human liver microsomes. This is done using large through-put studies with known substrates, or HPLC analyses of the metabolites. If inhibition of cytochrome P450 activity is detected, predictions of potential side effects can be made. Accurate measurement of inhibition can be performed in either in vivo or in vitro systems containing the specific cytochrome P450, a substrate and a putative inhibitor. Many cytochrome P450 enzymes are commercially available. Inhibitors of P450 enzymes are currently known, however, suitable N-dealkylase substrates for massive through-put studies are scarce.
Moody et al. (Xenobiotica 29: 53–75 (1999)) describe automated inhibition screens for the major human hepatic cytochromes P450. Radiometric analysis of erythromycin N-demethylation forg CYP3A4, dextromethorphan O-demethylation for CYP2D6, naproxen O-demethylation for CYP2C9 and diazepam N-demethylation for CYP2C19 were employed. For the radiometric assays greater than 99.7% of 14C-labelled substrate was routinely extracted from incubations by solid-phase extraction.
Stresser et al. (Drug Metabolism and Disposition 32:105–112 (2004)) tested azamulin, an anti-infective toward 18 different cytochromes P450 using human liver microsomes or microsomes from insect cells expressing single isoforms. The products from these chemical reactions were determined using HPLC analyses which are time consuming and labor intensive.